Tutor: Dott.ssa Tatiana Gianni (DIMES)
TITOLO DEL PROGETTO
Oncolytic Herpes Viruses retargeted to cancer-specific receptors: synergy between viral oncolysis and immune checkpoint blockade to boost the immune response against cancer
ABSTRACT DEL PROGETTO
To enter cell, human herpes simplex virus (HSV) use both viral fusion glycoproteins gH/gL/gB and viral glycoprotein gD that is responsible for cell/tissue tropism. Glycoprotein gD has been previously engineered with a single chain antibody specific for the HER2 receptor that is overexpressed by some types of cancer cells. The recombinant virus enters cells through the HER2 receptor only and has lost the ability to infect the cells that are the natural targets of HSV infection. This oncolytic, HER-2 retargeted virus (o-HSV-HER2ret) has been shown to be able to efficiently kill cancer cells both in vitro and in vivo models. However pre-clinical studies and clinical trials testing oncolytic viruses have demonstrated that, alone, the lytic activity of the virus against cancer cells is not sufficient for tumor clearance. An induction of an immune response is pivotal to give rise to a complete, immune-mediated tumor regression. The tumor micro-environment is immunosuppressive and cancer cells have devised strategies to evade the action of the immune system driving the upregulation of immune checkpoint molecules on the surface of cancer cells. For example, different types of tumors express PD-L1. PD-L1 binds to its receptor PD-1 expressed by activated T cells. As a result of these interactions, the T cell response is suppressed and the tumor escapes immune-mediated cell death. Other examples of immune checkpoint receptors are CTLA-4, LAG3 and TIM3. The systemic administration of monoclonal antibodies that block PD-1 and/or CTLA-4 to boost an immune response against cancer have been already approved for use as a treatment for some types of cancers. However systemic delivery of these blocking antibodies can cause severe adverse effects due to off target effects on the immune system. Local delivery of the antibodies inside the tumor microenvironment could drastically reduce the side effects. As such, we propose to insert in the o-HSV-HER2ret genome single chain antibodies that bind immune checkpoint molecules (PD-L1, CTLA-4, LAG3, TIM3) to restrict locally, in the cancer cells only, the expression of the immune checkpoint blockade. The rationale of using oncolytic viruses is to try to stimulate an initial immune response against tumor antigens. If the oncolytic virus is also engineered to express immune stimulatory molecules, the viral infection inside the tumor plus the aid of immune stimulation could be potent enough to overcome immune tolerance, leading to the development of an effective and long lasting immune response against cancer cells.
PIANO DELLE ATTIVITÀ DELL’ASSEGNISTA
- The gene encoding for a single chain antibody that recognizes mouse PD-L1 or CTLA-4 or PD-1 or LAG3, under a CMV promoter, will be inserted in the HSV US1-US2 intergenic region. An HSV BAC, which is retargeted to HER2 and carries the galK gene between US1 and US2 gene, is already available in the laboratory and it will be used as a starting point. The viruses will be regenerated by BAC transfection in SKOV3 cells. The supernatant harvested from the transfected SKOV3 will be tested by ELISA and by Western blot for the production of the single chain antibody of interest.
- The therapeutic potential and the ability of these o-HSVs to elicit a strong cancer-specific immune response will be tested in pre-clinical, immunocompetent mouse models of cancer carrying the human HER2 transgene. To develop these models, mouse cancer cell lines will be engineered to express human HER2 to allow entry of o-HSVs-HER2ret: a replication defective lentivirus carrying the human HER2 gene will be obtained by means of transfection of 293T cells with plasmids encoding lentivirus packaging proteins. This lentivirus will be used to transduced mouse cancer cells to obtain stable cell lines expressing human HER2. Different tumor cell lines will be generated to test the therapeutic potential of the o-HSVs against a wide range of cancer types.
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